Development and validation of an HPLC confirma‐tory method for the determination of tetracycline antibiotics residues in bovine muscle according to the European Union regulation 2002/657/EC

Abstract

A high-performance liquid chromatographic method with diode-array detection, at 351 nm, was developed and validated for the determination of five tetracyclines (TCs): minocycline, tetracycline, oxytetracycline, chlortetracycline, and doxycycline in bovine muscle. Samples were macerated with a buffer solution, centrifuged, and purified using Abselut Nexus SPE cartridges. The separation of the examined TCs was achieved on an Inertsil ODS-3 5 microm, 250 x 4 mm analytical column, at ambient temperature. A multistep gradient elution was followed using 0.05 M oxalic acid and CH3CN, at a flow rate of 1.65 mL/min. The procedure was validated according to the European Union regulation 2002/657/EC determining selectivity, stability, decision limit, detection capability, accuracy, and precision. The results of the validation process demonstrate that the method can be readily applied to European Union statutory veterinary drug residue surveillance programmes. Mean recoveries of TCs from bovine muscle samples spiked at three concentrations (100, 250, and 400 ng/g) were in the range of 98.7-103.3%. Method's LOQ values achieved were 40 microg/kg for MNC, CTC, and DC and 25 microg/kg for OTC and TC. The decision limits (CCalpha) were in the range of 104.7-109.8 microg/kg, while the detection capability (CCbeta) was in the range of 108.4-116.7 microg/kg for all compounds.

Development and validation of a method for the analysis of tetracyclines in chicken-muscle by liquid chromatography-electrospray-mass spectrometry in tandem (LC-ESI-MS/MS)

“…The first is defined as the limit above which it can be concluded, with an error probability of α, that a sample is noncompliant (higher than the MRL), and the second is defined as the smallest content of the substance that may be detected, identified, and/or quantified in a sample with an error probability of β. For these calculations, blank samples were fortified at MRL, extracted and analyzed; CCα and CCβ were calculated using the Equations 1 and 2: 19,24 CCa = yintercept + 1,64 x sintercept (1) slope CCb = yintercept + 1,64 x sintercept + 1;64 x sintercept (2) slope Stability of the analyte during storage is important since it may give rise to deviations in the outcome of the results of analysis. TC stability was determined in stock standard solution of 100 ng/mL prepared in methanol for each analyte.…”

“…The values for CCα and CCβ were in accordance to other studies of TCs in other food matrices. 21,24,34 …”

“…Under strongly acidic conditions anhydroTC are formed by a loss of water (H on C-5a, HO on C-6) and proton transfer (O-11/O-12), thereby extending the aromatic nature of the D ring to include the C ring. 9 Adequate treatment of samples is necessary to eliminate and/or reduce matrix interference, and is important to enrich the analyte of 9,14,[20][21][22][23][24] Several analytical procedures are available for the determination of TC antibiotics residues in animal tissues and chromatographic techniques are mainly used. High Performance Liquid Chromatography (HPLC) is a fast and reliable technique with high sensitivity for the analysis of TC antibiotics residues (identification, confirmation and quantification), varying its efficiency according to chromatographic conditions and type of detector used.…”

Termoestabilidad de sustancias antimicrobianas en la leche.

“…Como se observa en dicho Cuadro, muchos de los métodos empleados utilizan como "cleanup" una extracción en fase sólida antes del análisis cromatogáfico (Brandsteterová et al, 1997;Furusawa, 2003;Cinquina et al, 2003;Fritz et al, 2007;Samanidou et al, 2007). Sin embargo, otros autores han investigado sobre técnicas "cleanup" como la utilización de agentes quelantes y columnas MCAC (Metal Chelate Affinity) (Carson y Breslyn, 1996;Ruela et al, 2005), o el sistema MSPD de dispersión en fase sólida (Brandsteterová et al, 1997).…”

“…En otro estudio realizado por Samanidou et al (2007) sobre la validación de un método HPLC para la determinación de tetraciclinas en la leche, se indica que los extractos obtenidos y refrigerados a 4 ºC se mantuvieron estables únicamente una semana de acuerdo al criterio de aceptación indicado por este autor para la medición de la estabilidad de las sustancias antimicrobianas establecido en un -5% respecto a la concentración inicial. Estos resultados coinciden con los de este trabajo puesto que a los 7 días de refrigeración se obtienen, en los extractos refrigerados, pérdidas entre el 5,7 y 21,6%, correspondiendo con la pérdida de estabilidad establecida por dicho autor.…”